CN113881662A - Method for preparing single-stranded DNA (deoxyribonucleic acid) in solid-phase medium through 5' -terminal acrylamide-amidated PCR (polymerase chain reaction) product - Google Patents
Method for preparing single-stranded DNA (deoxyribonucleic acid) in solid-phase medium through 5' -terminal acrylamide-amidated PCR (polymerase chain reaction) product Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
Abstract
The invention discloses a method for preparing single-stranded DNA (deoxyribonucleic acid) in a solid-phase medium by using a 5' -terminal acrylamide PCR (polymerase chain reaction) product, belonging to the field of molecular biology. The preparation process comprises the following steps: (1) amplifying a large number of target fragments with 5' -terminal acrylamide through PCR reaction; (2) preparing a polyacrylamide gel solid phase separation medium; (3) dispersing the solid phase medium into fine particles by a high-shear emulsifying disperser in a high-speed homogenizing way; (4) and (3) performing pyrolysis and spiral on the DNA double strand, and separating a solid phase and a liquid phase to obtain the DNA single strand, namely the product. The method polymerizes a 5 '-acrylamido PCR product with polyacrylamide gel, binds the DNA double strand in a solid phase gel medium, unwinds through the high temperature DNA double strand, binds the 5' -acrylamido DNA strand in the polyacrylamide gel, and diffuses the other single strand in the supernatant. The single-stranded DNA prepared by the invention can be used in the fields of probe preparation and marking, solid-phase DNA sequencing, tumor microenvironment and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for preparing single-stranded DNA (deoxyribonucleic acid) in a solid-phase medium by using a PCR (polymerase chain reaction) product subjected to 5' -terminal acrylation.
Background
DNA plays an important role in numerous applications in bioimaging, biomimetic techniques and synthetic biology. Many of these applications rely on the availability of single stranded DNA. Depending on the size, scale and purity required, the production of single stranded DNA can become very expensive or burdensome. Most of DNA exists in a double helix structure, but becomes a single-stranded state upon heat or alkali treatment. The single-stranded DNA is different from the double-stranded DNA in molecular fluid mechanical properties, absorption spectrum, base reaction properties, and the like. Many fields of application require the use of single-stranded DNA templates. The single-stranded DNA templates obtained are useful for a variety of downstream applications such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry, pyrosequencing techniques and SNP analysis as well as single-stranded conformational polymorphisms, solid-phase DNA sequencing, DNA chips and microarrays, allele-specific extension and primer extension. Single-stranded DNA templates can also be used for in vitro mutagenesis, nuclease S1 localization, probe preparation and labeling, subtractive hybridization, and a variety of other molecular techniques. Methods for unwinding double strands are generally classified into chemical methods and physical methods. Chemical methods such as T7 reverse transcription, exonuclease, denaturing high performance liquid chromatography, magnetic bead capture, etc. are limited in that contamination with rnase must be strictly controlled, single strand yield depends on exonuclease activity, expensive instruments are required, and therefore, popularization is difficult, and coated magnetic beads used are expensive. On the other hand, although chip technology has been developed to provide a relatively simple and convenient method for large-scale synthesis of single-stranded DNA, most of them are limited by the kind of synthetic single-stranded DNA or experimental conditions, and cannot be generalized to become a routine laboratory method.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to overcome the limitations of expensive instruments, complex operation, difficult reagent acquisition and the like in the prior art and provide a method for preparing single-stranded DNA in a solid-phase medium by using a PCR product subjected to 5' -terminal acrylation. The preparation method can obtain single-stranded DNA in a conventional laboratory in a simple, effective and convenient way.
The invention is realized by the following technical scheme:
(1) amplifying a large number of target fragments with 5' terminal acrylamide by PCR reaction
Reaction systems for the control and experimental groups were prepared in 0.2ml PCR tubes according to the following table:
the reaction system prepared above was subjected to the following procedure on a PCR instrument:
(2) preparing a polyacrylamide gel solid phase separation medium, and washing for a plurality of times by using TE buffer solution or other buffer solutions to remove redundant primer, nucleic acid, buffer solution components and residual organic solvent in the gel preparation process;
| reagent | |
| 10× |
1× |
| 30％Acr-Bis(29∶1) | 7.5％Acr-Bis |
| 10% ammonium persulfate | 1.25％ |
| |
2％ |
| PCR reaction solution | 5uM |
| Water (W) | Is supplemented to 400ul |
Preparing non-denatured polyacrylamide gel with proper volume, wherein the system comprises PCR amplification reaction liquid which is carried out by a primer of 10 XTBE buffer solution, 10 percent ammonium persulfate, Tetramethylethylenediamine (TEMED), 30 percent Acr-Bis and 5' end modified acrylamide, so as to ensure that acrylamide monomers in the PCR reaction liquid are fully crosslinked with methylene bisacrylamide to form a gel solid phase medium.
The method for preparing the polyacrylamide gel solid phase medium is a system with a better concentration, and the method comprises the steps of sequentially adding 10 xTBE buffer solution, 30% Acr-Bis, a primer for modifying acrylamide at the 5' end, TEMED and 10% ammonium persulfate, and adding water to supplement the system to a proper system.
(3) Dispersing solid phase medium into fine particles by a high-shear emulsifying disperser in a high-speed homogenizing way, washing the fine particles for several times by using TE buffer solution or other buffer solutions to remove residual organic solvent, template nucleic acid molecules, 2 xTaq plus MasterMix, non-solid phase primers and the like;
(4) heating a magnetic stirrer, performing high-temperature denaturation and uncoiling of the DNA double strand, and separating a solid phase and a liquid phase to obtain a DNA single strand, namely a product.
Further, the 5' -acrylamido PCR product was crosslinked to polyacrylamide gel, and the double-stranded DNA was bound to a solid phase gel medium. By denaturing and uncoiling the DNA double strand at 80-100 deg.C, the 5' -acrylamido DNA single strand is bound to the polyacrylamide gel, while the other single strand of DNA diffuses into the supernatant.
Further, in the amplification reaction of the template, the 5' -terminal acrylamide-amidated primer is used as a solid phase primer, and is extended under the action of DNA polymerase, and the amplification reaction is carried out for 25-40 cycles, wherein the cycles comprise heating denaturation, annealing, and oligonucleotide primer extension under the action of 2 xTaq plus MasterMix and DNA polymerase.
Further, the DNA product is PCR amplification carried out by using a primer of which the 5' end modifies acrylamide, and reaction liquid is obtained; the primer sequence of the target band complementary strand is 5 'Acrydite-primer sequence-3', and the primer sequence of the target band is a normal sequence; the template is double-stranded DNA, and the reaction can be completed by the template and the specifically modified primer under the action of 2 xTaq plus MasterMix and DNA polymerase without other regeneration systems
Further, in the polymerization process of the PCR reaction solution and polyacrylamide gel, taking acrylamide as a unit, methylene bisacrylamide (Acr-Bis) is crosslinked to form a polymer with a linear polyacrylamide-co-acrylamide mark.
Further, binding the DNA double strand in a solid phase gel medium, heating at 80-100 ℃, magnetically stirring to denature the DNA double strand, and binding the 5' -acrylamide DNA single strand in polyacrylamide gel (precipitation), wherein the other single strand DNA is released and diffused in supernatant; separating solid phase and liquid phase, taking supernatant at 80-100 deg.C, and obtaining product with higher concentration by using high pressure vacuum concentrator; heating for more than 30min, stopping magnetic stirring, standing at high temperature for a period of time, observing the gel floc settling at the bottom with naked eyes, and slightly sucking appropriate amount of supernatant with a pipette to obtain the target product.
The primer sequences of the invention are as follows:
r primer-5 'Acrydite-primer sequence-3';
primer F-5 'primer sequence-3';
the verification and characterization of the product obtained in the step (4) comprise the following steps:
the ultraviolet spectrophotometer measures the absorbance difference at the specific wavelength of single-strand and double-strand DNA, and the absorbance of G \ C double bonds at A260 is larger because the base of the single-strand DNA is exposed more than that of the double-strand DNA.
The content of the ultra-trace nucleic acid protein is detected by an ultra-trace nucleic acid protein detector, A260 is the maximum absorption wavelength of nucleic acid, A280 is the maximum absorption wavelength of protein, the ratio of the two reflects the purity of the sample, and A260/A280 of the pure DNA sample is more than 1.8.
8% native polyacrylamide gel electrophoresis verification experiment, because double strand DNA charge density is bigger, topological structure is tighter, and covalently closed DNA steric hindrance is smaller, and open-loop DNA steric hindrance is bigger, so double strand DNA runs faster than single strand DNA in the electrophoresis chart.
The invention has the characteristics obviously superior to the prior art, and has the positive progress effects that:
(1) the cost is reduced, and the complicated operation process is avoided: synthesizing a specific target fragment by using a conventional primer, polymerizing the specific target fragment with polyacrylamide gel, dispersing the specific target fragment into fine particles, washing, and thermally denaturing and uncoiling the fine particles under physical conditions to obtain the polyacrylamide gel;
(2) the novelty is as follows: the invention establishes a method for heating and releasing double chains in vitro based on the conventional experimental PCR reaction and polyacrylamide gel, and does not need other reagents such as specific shear enzyme, restriction endonuclease and the like;
(3) simple and easy to obtain: the reagent used in the invention is a PCR reaction system and a reagent for preparing polyacrylamide gel in a conventional laboratory, and has no other special characteristics; the target product can be obtained only by heating under physical conditions without complex chemical processes.
Drawings
FIG. 1 is a schematic flow chart of a method for preparing single-stranded DNA. Wherein (a) is a PCR reaction of a 5' -acrylamido primer; (b) is formed by polyacrylamide gel and separated from the target product;
FIG. 2 is a comparison of 5' -acrylamido primers reacted with unmodified primers by PCR and polyacrylamide gel polymerization;
FIG. 3 shows the absorbance of DNA at A260 by UV spectrophotometry;
FIG. 4 is a DNA electrophoresis chart of 8% non-denaturing polyacrylamide gel;
FIG. 5 is a graph of nucleic acid dye method versus single-stranded DNA characterization;
wherein M represents a marker, the marker is a double-chain marker in a polyacrylamide gel electrophoresis result picture, and the ssDNA and the dsDNA with the same length have slower migration rate in the polyacrylamide gel electrophoresis. F-Acr 1 represents that the GoldView I type nucleic acid dye dyes acrylamide single-stranded DNA; F-Acr2 shows staining of acrylamido single stranded DNA with a GoldView type II nucleic acid dye.
Detailed Description
The invention is further illustrated by way of example in the following description and drawings. The invention is not to be thus limited in scope by the examples described. The experimental methods without specifying the specific conditions in the examples below were selected according to the conventional methods and conditions, or according to the commercial specifications.
Based on the limitation existing in the prior art and the wide application of single-stranded DNA, the invention provides a novel research method for quickly obtaining the single-stranded DNA in vitro in a simple, effective and convenient way, and the length of the obtained single-stranded DNA is about 400 nt. The invention firstly uses 5 'acrylate modified primer and DNA template to make PCR amplification, then makes PCR amplification product and acrylamide, 10% ammonium persulfate and tetramethylethylenediamine implement copolymerization to form polymer with linear polyacrylamide-co-acrylic acid label, and makes said copolymer undergo the processes of high-speed homogeneous breaking, and makes thermal denaturation at high temperature of 94 deg.C, the 5' -acrylate modified synthetic chain is bound in polyacrylamide gel (precipitation), another chain is dispersed in the solution, and utilizes the centrifugation or standing still for a period of time to obtain supernatant liquor, and utilizes high-pressure vacuum concentrator to obtain product with higher concentration, and its preparation process diagram is shown in figure 1
Example 1: a5' -terminal acrylamido single-stranded DNA was prepared and characterized.
GTTTCAATTCGTACAATGCCTGGCATGTTCATTCGAATATAAGGCCGCCGCCTTCCA GTCAGGGTAGCCAAAAGTATAATCCCGGGTGGAAACTAAACTAAAAACCGTACTCAC AACTTTCCGCGGACGCTAACAGACAAATAGACACACTATCAGGTCAGGAACTGCCGT CACATACGACACTGCCCCTCACGTAAGGGCCACCGACCATGTGGGCAAATTCGTAAT AAATTCGGGGTGAGGGGGATTCAAGACAAGCAACCTTGTTAGTCAGCTCAAACAGCG ATTTAACGGTTGAGTAACACATCAAAACACCGTTCGAGGTCAAGCCTGGCGTGTTTA ACAAGTTCTTGATATCATATATAAATGTAATAAGAAGTTTGGTAATATTCAATTCGA AG (shown in SEQ ID No. 1)
R primer: 5 'Acrydite-CTTCGAATTGAATACCAAACTT-3' (shown in SEQ ID No. 2)
F primer: 5'-GTTTCAATTCGTACAATGCCTGGCATG-3' (shown in SEQ ID No. 3). The corresponding double-stranded DNA was amplified by 5' -end acrylamide primer as shown in FIG. 1 (a).
Reaction systems for the control and experimental groups were prepared in 0.2ml PCR tubes according to the following table:
| 50uL reaction system | |
||
| 2× | 25uL | 1× | |
| 10uM 5' Acrydite-R primer | 2uL | 0.4uM | |
| 10uM F primer | 2uL | 0.4uM | |
| Form panel | 2uL | 25ng | |
| Water (W) | 19uL |
The reaction system prepared above was subjected to the following procedure on a PCR instrument:
a gel polymerization system was prepared according to the following table:
| reagent | Volume (uL) |
| 10×TBE | 40 |
| 30％Acr-Bis(29∶1) | 100 |
| 10% ammonium persulfate | 3.2 |
| Tetramethyl ethylene diamine | 0.4 |
| PCR reaction solution | 200 |
| Water (W) | 56.4 |
Results of the reaction
As shown in FIG. 2, when nucleic acid dyes were added to the control and experimental groups, respectively, it was observed that the target fragment amplified with the 5' Acrydite-R primer was firmly embedded in the polyacrylamide gel under the gel imager, while no fluorescence was observed in the gel from the reaction solution amplified with the unmodified primer.
Crushing the gel into floccule, heating at 94 deg.C for 30min, standing at high temperature, collecting supernatant, and measuring absorbance and content.
As shown in FIG. 3, the UV spectrophotometer detects the difference between the absorbance of ssDNA and dsDNA at A260, and the result shows that the absorbance of ssDNA at A260 is larger than that of dsDNA, and the difference has statistical significance (P < 0.0001), which is in line with the expected result of the target product.
As shown in Table 1, the content and purity of ssDNA detected by the ultra-trace nucleic acid protein detector showed that the ratio of ssDNA to dsDNA was higher in A260/A280, which is consistent with the expected results.
TABLE 1
As shown in FIG. 4, 8% native polyacrylamide gel DNA electrophoresis (100V voltage, 1h) verifies the target product and dsDNA, and the result shows that the ssDNA is positioned forward relative to the dsDNA, i.e., the migration rate is slower relative to the dsDNA, and no other obvious band appears in the electrophoresis image, which is expected.
Example 2: the F primer is modified by acrylamide to prepare single-stranded DNA.
R primer: 5'-CTTCGAATTGAATACCAAACTT-3'
F primer: 5 'Acrydite-GTTTCAATTCGTACAATGCCTGGCATG-3'
Reaction systems for the control and experimental groups were prepared in 0.2ml PCR tubes according to the following table:
| 20uL reaction system | |
|
| 2×T5 Super PCR Mix(PAGE) | |
1× |
| 10uM R primer | 1uL | 0.5uM |
| 10uM 5' Acrydite-F primer | 1uL | 0.5uM |
| Form panel | 1uL | 1ug |
| Water (W) | 7uL |
The reaction system prepared above was subjected to the following procedure on a PCR instrument:
a gel polymerization system was prepared according to the following table:
| reagent | Volume (uL) |
| 10×TBE | 80 |
| 30％Acr-Bis(29∶1) | 200 |
| 10% ammonium persulfate | 6.4 |
| Tetramethyl ethylene diamine | 0.8 |
| PCR reaction solution | 400 |
| Water (W) | 112.8 |
Crushing the gel into floccule, heating at 94 ℃ for 30min, standing at high temperature, taking supernatant, and characterizing by a nucleic acid dye method.
Results of the reaction
By adding GoldView type I (aiming at double-stranded DNA) and type II (aiming at single-stranded DNA) nucleic acid dyes into the prepared single-strand and PCR reaction liquid before heating for characterization, a bright band can be observed by carrying out electrophoresis after mixing the PCR reaction substance before heating and the GoldView type I nucleic acid dyes; the single-stranded DNA was mixed with the GoldView type I nucleic acid dye and then electrophoretically observed without bands, while the mixture with the GoldView type II nucleic acid dye and then electrophoretically observed with bands, indicating that the single-stranded DNA was prepared by the above method.
As shown in FIG. 5, 8% native polyacrylamide gel DNA electrophoresis (100V voltage, 1h) confirmed the target product and dsDNA, and the results showed that the nucleic acid dye GoldView I did not develop ssDNA and the nucleic acid dye GoldView II developed ssDNA, as expected. The above-described examples are given for clarity of illustration only and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. The invention has not been described in detail in order to avoid obscuring the invention.
Sequence listing
<110> university of teachers in Hangzhou
<120> a method for preparing single-stranded DNA in a solid medium by 5' -terminal acrylamidization of PCR product
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gtttcaattc gtacaatgcc tggcatgttc attcgaatat aaggccgccg ccttccagtc 60
agggtagcca aaagtataat cccgggtgga aactaaacta aaaaccgtac tcacaacttt 120
ccgcggacgc taacagacaa atagacacac tatcaggtca ggaactgccg tcacatacga 180
cactgcccct cacgtaaggg ccaccgacca tgtgggcaaa ttcgtaataa attcggggtg 240
agggggattc aagacaagca accttgttag tcagctcaaa cagcgattta acggttgagt 300
aacacatcaa aacaccgttc gaggtcaagc ctggcgtgtt taacaagttc ttgatatcat 360
atataaatgt aataagaagt ttggtaatat tcaattcgaa g 401
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gtttcaattc gtacaatgcc tggcatg 27
Claims (5)
1. A method for preparing single-stranded DNA in a solid medium by 5' -terminal acrylamidization of a PCR product, comprising the steps of:
1) preparing a PCR reaction system, amplifying a large number of target fragments with 5 '-terminal acrylamide through PCR reaction, taking a template, taking a primer with 5' -terminal acrylamide as a solid phase primer in the amplification reaction of the template, extending under the action of DNA polymerase, and carrying out PCR amplification on the reaction system;
2) carrying out gel making treatment on the PCR reaction solution prepared in the step 1), preparing a polyacrylamide gel solid phase separation medium, and washing for a plurality of times by using a TE buffer solution to remove redundant primer, nucleic acid, buffer solution components and residual organic solvent in the gel making process;
3) adding the gel prepared in the step 2) into a buffer solution, uniformly dispersing a solid phase medium into fine particles at a high speed by a high-shear emulsification dispersion machine, washing the fine particles for several times by using the buffer solution to remove residual organic solvent, template nucleic acid molecules, 2 xTaq plus MasterMix and non-solid phase primers, and preparing a suspoemulsion;
4) placing the suspension emulsion prepared in the step 3) under a heating magnetic stirrer, performing high-temperature denaturation and uncoiling on the DNA double strand, and separating a solid phase and a liquid phase to obtain a DNA single strand, namely a single-strand DNA product.
2. The method for preparing single-stranded DNA in a solid medium by 5' -terminal acrylamidization of PCR product as set forth in claim 1, wherein the PCR reaction system in step 1) comprises: 2 × Taq plusMasterMix final concentration 1 ×; 10uM 5' of R primer, final concentration 0.2-1.0 uM; 10uM F primer, final concentration 0.2-1.0 uM; template, plasmid at a final concentration of 1-50 ng and a base at a final concentration of 10 ng-1 ugGrouping the genes; ddH2O was supplemented to 50 uL.
3. The method for preparing single-stranded DNA in solid medium by 5' -terminal acrylamidization of PCR product as claimed in claim 1, wherein the PCR reaction procedure in step 1) is: pre-denaturation: 94 ℃, 5min, cycle number 1; denaturation: 30s at 94 ℃; annealing: tm-5 ℃ for 30 s; extension: 72 ℃, 1 kb/min; the number of cycles of denaturation, annealing and extension is 25-40; final extension, 72 ℃, cycle number 1.
4. The method for preparing single-stranded DNA in solid phase medium by 5 '-terminal acrylamide amidation of PCR product of claim 1, wherein the amine gel solid phase separation medium system of step 2) comprises PCR amplification reaction solution of 10 XTBE buffer, 10% ammonium persulfate, Tetramethylethylenediamine (TEMED), 30% Acr-Bis, and 5' -terminal acrylamide modified primer, so as to ensure that acrylamide monomer in the PCR reaction solution is fully crosslinked with methylene bisacrylamide to form gel solid phase medium.
5. The method for preparing single-stranded DNA in solid medium by 5' -terminal acrylamidization of PCR product as set forth in claim 1, wherein the reaction conditions in step 4) are: heating at 80-100 deg.C and magnetically stirring for at least 30min, separating solid/liquid phase at 80-100 deg.C to obtain DNA single strand, and concentrating for more than 2 hr with high pressure vacuum concentrator to obtain product with higher concentration.
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|---|---|---|---|---|
| CN1733935A (en) * | 2005-06-17 | 2006-02-15 | 东南大学 | Preparation method of DNA microarray chip based on gel fixed nucleic acid |
| KR20160056195A (en) * | 2014-11-11 | 2016-05-19 | 충북대학교 산학협력단 | Amplification apparatus and method of Single Strand DNA using Microbeads |
| US20200190505A1 (en) * | 2016-09-12 | 2020-06-18 | Dana-Farber Cancer Institute, Inc. | Acrylamide copolymerization for sequestration and production of single-stranded nucleic acid |
| US20210032683A1 (en) * | 2018-04-12 | 2021-02-04 | Dana-Farber Cancer Institute, Inc. | Dna-tagged methanol responsive polymer for single-stranded nucleic acid production |
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| CN1733935A (en) * | 2005-06-17 | 2006-02-15 | 东南大学 | Preparation method of DNA microarray chip based on gel fixed nucleic acid |
| KR20160056195A (en) * | 2014-11-11 | 2016-05-19 | 충북대학교 산학협력단 | Amplification apparatus and method of Single Strand DNA using Microbeads |
| US20200190505A1 (en) * | 2016-09-12 | 2020-06-18 | Dana-Farber Cancer Institute, Inc. | Acrylamide copolymerization for sequestration and production of single-stranded nucleic acid |
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